Tong Qiang-Song, Zheng Li-Duan, Tang Shao-Tao, Jiang Guo-Song, Ruan Qing-Lan, Zeng Fu-Qing, Dong Ji-Hua
Department of Surgery, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Acta Pharmacol Sin. 2007 May;28(5):672-84. doi: 10.1111/j.1745-7254.2007.00552.x.
To characterize the molecular mechanisms of nitrofen-induced pulmonary hypoplasia.
After administration of nitrofen to cultured type II A549 pneumocytes, cell proliferation and DNA synthesis were investigated by 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide colorimetry, colony formation assay, flow cytometry and [3H]-thymidine incorporation assay. Apoptosis was measured by terminal transferase-mediated dUTP nick-end-labeling, acridine orange-ethidium bromide staining and flow cytometry. Expression of proliferating cell nuclear antigen (PCNA) and apoptosis-related genes was assayed by immunofluorescence, RT-PCR and Western blot.
Nitrofen inhibited the cell proliferation of A549 cells in a dose- and time-dependent manner, accompanied by downregulation of PCNA. As a result, the DNA synthesis of nitrofentreated A549 cells decreased, while cell cycle was arrested at G0/G1 phase. Moreover, nitrofen induced apoptosis of A549 cells, which was not abolished by Z-Val-Ala- Asp(OCH3)- fluoromethylketone. In addition, nitrofen decreased the expression of Bcl-x( L), but not of Bcl-2, Bax, and Bak, resulting in a loss of mitochondrial membrane potential and the nuclear translocation of apoptosis-inducing factor (AIF). Meanwhile, nitrofen strongly activated the p38 mitogen-activated protein kinase (p38-MAPK). Pretreatment of cells with SB203580 (5 micromol/L) blocked nitrofen-induced phosphorylation of p38-MAPK and abolished nitrofen-induced AIF translocation and apoptosis in A549 cells.
Nitrofen suppresses the proliferation of cultured type II pneumocytes accompanied by the downregulation of PCNA, and induces mitochondria-mediated apoptosis involving the activation of p38-MAPK.
阐明除草醚诱导肺发育不全的分子机制。
将除草醚作用于培养的II型A549肺细胞后，采用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐比色法、集落形成试验、流式细胞术和[3H]-胸腺嘧啶核苷掺入试验研究细胞增殖和DNA合成。通过末端脱氧核苷酸转移酶介导的dUTP缺口末端标记、吖啶橙-溴化乙锭染色和流式细胞术检测细胞凋亡。采用免疫荧光、逆转录-聚合酶链反应和蛋白质印迹法检测增殖细胞核抗原（PCNA）和凋亡相关基因的表达。
除草醚以剂量和时间依赖性方式抑制A549细胞的增殖，同时PCNA表达下调。结果，经除草醚处理的A549细胞的DNA合成减少，而细胞周期停滞于G0/G1期。此外，除草醚诱导A549细胞凋亡，而Z-缬氨酸-丙氨酸-天冬氨酸(甲酯)-氟甲基酮不能消除这种凋亡。另外，除草醚降低Bcl-x(L)的表达，但不影响Bcl-2、Bax和Bak的表达，导致线粒体膜电位丧失和凋亡诱导因子（AIF）的核转位。同时，除草醚强烈激活p38丝裂原活化蛋白激酶（p38-MAPK）。用SB203580（5 μmol/L）预处理细胞可阻断除草醚诱导的p38-MAPK磷酸化，并消除除草醚诱导的A549细胞AIF转位和凋亡。
除草醚抑制培养的II型肺细胞增殖，同时下调PCNA，并通过激活p38-MAPK诱导线粒体介导的细胞凋亡。
